Weak acids are widely used as food preservatives, as they are very effective at preventing the growth of most species of bacteria and fungi. However, some species of molds can survive and grow in the concentrations of weak acid employed in food and drink products, thereby causing spoilage with resultant risks for food security and health. Current knowledge of weak-acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. We characterized gene functions in the spoilage mold species Aspergillus niger which are important for survival and growth in the presence of weak-acid preservatives. Such identification of weak-acid resistance mechanisms in spoilage molds will help in the design of new strategies to reduce food spoilage in the future.

Spoilage by yeasts and molds is a major contributor to food and drink waste, which undermines food security. Weak acid preservatives like sorbic acid help to stop spoilage, but some yeasts, commonly associated with spoilage, are resistant to sorbic acid. Different yeasts generate energy for growth by the processes of respiration and/or fermentation. Here, we show that sorbic acid targets the process of respiration, so fermenting yeasts are more resistant. Fermentative yeasts are also those usually found in spoilage incidents. This insight helps to explain the spoilage of sorbic acid-preserved foods by yeasts and can inform new strategies for effective control. This is timely as the sugar content of products like soft drinks is being lowered, which may favor respiration over fermentation in key spoilage yeasts.

Osmotolerance or halotolerance are used to describe resistance to sugars and salt, or only salt, respectively. Here, a comprehensive screen of more than 600 different yeast isolates revealed that osmosensitive species were equally affected by NaCl and glucose. However, the relative toxicity of salt became increasingly prominent in more osmoresistant species. We confirmed that growth inhibition by glucose in a laboratory strain of Saccharomyces cerevisiae occurred at a lower water activity (Aw) than by salt (NaCl), and pre-growth in high levels of glucose or salt gave enhanced cross-resistance to either. Salt toxicity was largely due to osmotic stress but with an additive enhancement due to effects of the relevant cation. Almost all of the yeast isolates from the screen were also noted to exhibit hetero-resistance to both salt and sugar, whereby high concentrations restricted growth to a small minority of cells within the clonal populations. Rare resistant colonies required growth for up to 28 days to become visible. This cell individuality was more marked with salt than sugar, a possible further reflection of the ion toxicity effect. In both cases, heteroresistance in S. cerevisiae was strikingly dependent on the GPD1 gene product, important for glycerol synthesis. In contrast, a tps1Δ deletant impaired for trehalose showed altered MIC but no change in heteroresistance. Effects on heteroresistance were evident in chronic (but not acute) salt or glucose stress, particularly relevant to growth on low Aw foods. The study reports diverse osmotolerance and halotolerance phenotypes and heteroresistance across an extensive panel of yeast isolates, and indicates that Gpd1-dependent glycerol synthesis is a key determinant enabling growth of rare yeast subpopulations at low Aw, brought about by glucose and in particular salt.

Introduction: CXD101 is a novel class I-selective HDAC inhibitor (HDACi). It is specific to class I HDAC1 isoforms and has no activity against class II HDAC. CXD101 induces specific HDAC inhibition and enhanced histone acetylation in a time and dose-dependent fashion in vitro, in treated canine and murine leucocytes in vivo, and in human leucocytes treated ex vivo. CXD101 has an improved pharmacokinetic (PK) profile compared to other HDACi in pre-clinical studies. Methods: We determined the maximal tolerated dose (MTD) of CXD101 in a dose escalation study in advanced cancer. The dose was escalated from 1 mg bd for 5 days (d) in a 21D cycle (C), following a 3 + 3 design. A preliminary efficacy analysis was performed. A total of 39 pts were enrolled; 36 were exposed to CXD101. Results: The MTD and recommended phase II dose (RP2D) was 20 mg bd. In 17 lymphoma pts dosed at 316mg bd, 3 had partial responses (2 Hodgkin lymphoma post allograft, 1 refractory angioimmunoblastic T-cell lymphoma) and 1 complete response (relapsed follicular lymphoma) (overall response 23.5%) alongside 7 pts gaining benefit with stable disease. Responses were durable (203d, 161d, 141d (ongoing), and 176d (ongoing), respectively). Tumour volume reduction was seen in 56%. The frequent adverse effects (AEs) were fatigue, nausea and cytopenias. CXD101 was well tolerated and all AEs were manageable. CXD101 showed considerable variation in uptake, but peak concentrations were reached at 1–4 hours (h) post dosing (mean time to peak on d5: 2.3 ± 1.1 h). The mean PK data for C1 and 2 are shown (Table 1). Even at 25 mg b.d., there was no detectable drug at the next d1. T1/2 was 5-12 h, with a longer t1/2 on d5, C1 (d1 6.8 ± 3.8 h, d5 10.2 ± 4.3 h). CXD101 did not accumulate; the ratio between the area under the curve (AUC) on d1 and d5 was 2–3, with a mean of 2.86 ± 0.94. There was a linear relationship between dose and AUC, Cmax and Cmin (8 h post-dose) for both cycles and days (shown C1 d5: Fig 1A–C), although there was some inter-individual PK parameter variation (R2 coefficient 0.86–0.91). The mean accumulation ratio was consistent with a terminal t1/2 of 19.5 h. The average Cmax to Cmin at RP2D ranged 300–200 nM, well within the biologically active range in vitro. Conclusion: The peak concentration was at 1–4 h, t1/2 was 5–12 h, with little accumulation with multiple cycles. There was a linear relationship between CXD101 dose and AUC, Cmaxand Cmin. CXD101 has encouraging, durable activity in Hodgkin lymphoma, T-cell lymphoma, and follicular lymphoma in the early stages of its development. Keywords: histone deacetylase inhibitors.

The early stages of development of Aspergillus niger conidia during outgrowth were explored by combining genome-wide gene expression analysis (RNAseq), proteomics, Warburg manometry and uptake studies. Resting conidia suspended in water were demonstrated for the first time to be metabolically active as low levels of oxygen uptake and the generation of carbon dioxide were detected, suggesting that low-level respiratory metabolism occurs in conidia for maintenance. Upon triggering of spore germination, generation of CO2 increased dramatically. For a short period, which coincided with mobilisation of the intracellular polyol, trehalose, there was no increase in uptake of O2 indicating that trehalose was metabolised by fermentation. Data from genome-wide mRNA profiling showed the presence of transcripts associated with fermentative and respiratory metabolism in resting conidia. Following triggering of conidial outgrowth, there was a clear switch to respiration after 25min, confirmed by cyanide inhibition. No effect of SHAM, salicylhydroxamic acid, on respiration suggests electron flow via cytochrome c oxidase. Glucose entry into spores was not detectable before 1h after triggering germination. The impact of sorbic acid on germination was examined and we showed that it inhibits glucose uptake. O2 uptake was also inhibited, delaying the onset of respiration and extending the period of fermentation. In conclusion, we show that conidia suspended in water are not completely dormant and that conidial outgrowth involves fermentative metabolism that precedes respiration.

The genus Dekkera/Brettanomyces comprises five described species: Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. naardenensis and B. nanus. Some of them, especially D. bruxellensis, are important spoilage organisms, particularly in the wine and beverage industries. Because of their economic importance many different methods have been developed to identify members of the genus in general and D. bruxellensis in particular. These methods vary in their rapidity, complexity and cost but, partly because of confidentiality issues, it is unclear which methods are used, or how widely, in the relevant industries. Building on previous work with the genera Saccharomyces and Zygosaccharomyces, a suite of eight PCR primer pairs has been designed either on the D1-D2 region of the 26S rRNA gene or translation elongation factor TEF1-α. These primers can specifically identify the genus as a whole, only Dekkera species, each one of the five recognised species as well as a significant subgroup of D. bruxellensis represented by NCYC 3426. Multiplexing has also been tried and it has been shown to be possible with some combinations of genus or Dekkera-level and species-specific primers. Using direct colony PCR amplification followed by gel electrophoresis, a clear positive result can be obtained in less than 3h, thus providing a quick, reliable and inexpensive way to identify target species.

ABSTRACT Conidial germination is fundamentally important to the growth and dissemination of most fungi. It has been previously shown (K. Hayer, M. Stratford, and D. B. Archer, Appl. Environ. Microbiol. 79:6924–6931, 2013, http://dx.doi.org/10.1128/AEM.02061-13 ), using sugar analogs, that germination is a 2-stage process involving triggering of germination and then nutrient uptake for hyphal outgrowth. In the present study, we tested this 2-stage germination process using a series of nitrogen-containing compounds for the ability to trigger the breaking of dormancy of Aspergillus niger conidia and then to support the formation of hyphae by acting as nitrogen sources. Triggering and germination were also compared between A. niger and Aspergillus nidulans using 2-deoxy- d -glucose (trigger), d -galactose (nontrigger in A. niger but trigger in A. nidulans ), and an N source (required in A. niger but not in A. nidulans ). Although most of the nitrogen compounds studied served as nitrogen sources for growth, only some nitrogen compounds could trigger germination of A. niger conidia, and all were related to l -amino acids. Using l -amino acid analogs without either the amine or the carboxylic acid group revealed that both the amine and carboxylic acid groups were essential for an l -amino acid to serve as a trigger molecule. Generally, conidia were able to sense and recognize nitrogen compounds that fitted into a specific size range. There was no evidence of uptake of either triggering or nontriggering compounds over the first 90 min of A. niger conidial germination, suggesting that the germination trigger sensors are not located within the spore.

The food spoilage yeast Zygosaccharomyces bailii shows great resistance to weak-acid preservatives, including sorbic acid (2, 4-hexadienoic acid). That extreme resistance was shown to be due to population heterogeneity, with a small sub-population of cells resistant to a variety of weak acids, probably caused by a lower internal pH reducing the uptake of all weak acids. In the present paper, it was found that resistant cells were extremely rare in exponential cultures, but increased by up to 8000-fold in stationary phase. Inoculation of media containing sorbic acid with a population of Z. bailii cells gave rise to what appeared to be a prolonged lag phase, suggesting adaptation to the conditions before the cells entered the period of exponential growth. However, the apparent lag phase caused by sorbic acid was largely due to the time required for the resistant sub-population to grow to detectable levels. The slow growth rate of the sub-population was identical to that of the final total population. The non-resistant bulk population remained viable for 3days but had lost viability by 6days and, during that time, there was no indication of any development of resistance in the bulk population. The sub-population growing in sorbic acid showed very high population diversity in colony size and internal pH. After removal of sorbic acid, the population rapidly reverted back to the normal, largely non-resistant, population distribution. The data presented suggest that a reevaluation of the lag phase in microbial batch culture is required, at least for the resistance of Z. bailii to sorbic acid. Furthermore, the significance of phenotypic diversity and heterogeneity in microbial populations is discussed more broadly with potential relevance to bacterial “persisters”, natural selection and evolution.

Weak-acid preservatives, such as sorbic acid and acetic acid, are used in many low pH foods to prevent spoilage by fungi. The spoilage yeast Zygosaccharomyces bailii is notorious for its extreme resistance to preservatives and ability to grow in excess of legally-permitted concentrations of preservatives. Extreme resistance was confirmed in 38 strains of Z. bailii to several weak-acid preservatives. Using the brewing yeast Saccharomyces cerevisiae as a control, tests showed that Z. bailii was ~ 3-fold more resistant to a variety of weak-acids but was not more resistant to alcohols, aldehydes, esters, ethers, ketones, or hydrophilic chelating acids. The weak acids were chemically very diverse in structure, making it improbable that the universal resistance was caused by degradation or metabolism. Examination of Z. bailii cell populations showed that extreme resistance to sorbic acid, benzoic acid and acetic acid was limited to a few cells within the population, numbers decreasing with concentration of weak acid to < 1 in 1000. Re-inoculation of resistant sub-populations into weak-acid-containing media showed that all cells now possessed extreme resistance. Resistant sub-populations grown in any weak-acid preservative also showed ~ 100% cross-resistance to other weak-acid preservatives. Tests using 14C-acetic acid showed that weak-acid accumulation was much lower in the resistant sub-populations. Acid accumulation is caused by acid dissociation in the higher pH of the cytoplasm. Tests on intracellular pH (pHi) in the resistant sub-population showed that the pH was much lower, ~ pH 5.6, than in the sensitive bulk population. The hypothesis is proposed that extreme resistance to weak-acid preservatives in Z. bailii is due to population heterogeneity, with a small proportion of cells having a lower intracellular pH. This reduces the level of accumulation of any weak acid in the cytoplasm, thus conferring resistance to all weak acids, but not to other inhibitors.

BACKGROUND:Genome-wide analysis was performed to assess the transcriptional landscape of germinating A. niger conidia using both next generation RNA-sequencing and GeneChips. The metabolism of storage compounds during conidial germination was also examined and compared to the transcript levels from associated genes.RESULTS:The transcriptome of dormant conidia was shown to be highly differentiated from that of germinating conidia and major changes in response to environmental shift occurred within the first hour of germination. The breaking of dormancy was associated with increased transcript levels of genes involved in the biosynthesis of proteins, RNA turnover and respiratory metabolism. Increased transcript levels of genes involved in metabolism of nitrate at the onset of germination implies its use as a source of nitrogen. The transcriptome of dormant conidia contained a significant component of antisense transcripts that changed during germination.CONCLUSION:Dormant conidia contained transcripts of genes involved in fermentation, gluconeogenesis and the glyoxylate cycle. The presence of such transcripts in dormant conidia may indicate the generation of energy from non-carbohydrate substrates during starvation-induced conidiation or for maintenance purposes during dormancy. The immediate onset of metabolism of internal storage compounds after the onset of germination, and the presence of transcripts of relevant genes, suggest that conidia are primed for the onset of germination. For some genes, antisense transcription is regulated in the transition from resting conidia to fully active germinants.

The asexual spores (conidia) of Aspergillus niger germinate to produce hyphae under appropriate conditions. Germination is initiated by conidial swelling and mobilization of internal carbon and energy stores, followed by polarization and emergence of a hyphal germ tube. The effects of different pyranose sugars, all analogues of d-glucose, on the germination of A. niger conidia were explored, and we define germination as the transition from a dormant conidium into a germling. Within germination, we distinguish two distinct stages, the initial swelling of the conidium and subsequent polarized growth. The stage of conidial swelling requires a germination trigger, which we define as a compound that is sensed by the conidium and which leads to catabolism of d-trehalose and isotropic growth. Sugars that triggered germination and outgrowth included d-glucose, d-mannose, and d-xylose. Sugars that triggered germination but did not support subsequent outgrowth included d-tagatose, d-lyxose, and 2-deoxy-d-glucose. Nontriggering sugars included d-galactose, l-glucose, and d-arabinose. Certain nontriggering sugars, including d-galactose, supported outgrowth if added in the presence of a complementary triggering sugar. This division of functions indicates that sugars are involved in two separate events in germination, triggering and subsequent outgrowth, and the structural features of sugars that support each, both, or none of these events are discussed. We also present data on the uptake of sugars during the germination process and discuss possible mechanisms of triggering in the absence of apparent sugar uptake during the initial swelling of conidia.

Weak-acid preservatives commonly used to prevent fungal spoilage of low pH foods include sorbic and acetic acids. The “classical weak-acid theory” proposes that weak acids inhibit spoilage organisms by diffusion of undissociated acids through the membrane, dissociation within the cell to protons and anions, and consequent acidification of the cytoplasm. Results from 25 strains of Saccharomyces cerevisiae confirmed inhibition by acetic acid at a molar concentration 42 times higher than sorbic acid, in contradiction of the weak-acid theory where all acids of equal pKa should inhibit at equimolar concentrations. Flow cytometry showed that the intracellular pH fell to pH 4.7 at the growth-inhibitory concentration of acetic acid, whereas at the inhibitory concentration of sorbic acid, the pH only fell to pH 6.3. The plasma membrane H+-ATPase proton pump (Pma1p) was strongly inhibited by sorbic acid at the growth-inhibitory concentration, but was stimulated by acetic acid. The H+-ATPase was also inhibited by lower sorbic acid concentrations, but later showed recovery and elevated activity if the sorbic acid was removed. Levels of PMA1 transcripts increased briefly following sorbic acid addition, but soon returned to normal levels. It was concluded that acetic acid inhibition of S. cerevisiae was due to intracellular acidification, in accord with the “classical weak-acid theory”. Sorbic acid, however, appeared to be a membrane-active antimicrobial compound, with the plasma membrane H+-ATPase proton pump being a primary target of inhibition. Understanding the mechanism of action of sorbic acid will hopefully lead to improved methods of food preservation.

Moulds are able to cause spoilage in preserved foods through degradation of the preservatives using the Pad-decarboxylation system. This causes, for example, decarboxylation of the preservative sorbic acid to 1,3-pentadiene, a volatile compound with a kerosene-like odour. Neither the natural role of this system nor the range of potential substrates has yet been reported. The Pad-decarboxylation system, encoded by a gene cluster in germinating spores of the mould Aspergillus niger, involves activity by two decarboxylases, PadA1 and OhbA1, and a regulator, SdrA, acting pleiotropically on sorbic acid and cinnamic acid. The structural features of compounds important for the induction of Pad-decarboxylation at both transcriptional and functionality levels were investigated by rtPCR and GCMS. Sorbic and cinnamic acids served as transcriptional inducers but ferulic, coumaric and hexanoic acids did not. 2,3,4,5,6-Pentafluorocinnamic acid was a substrate for the enzyme but had no inducer function; it was used to distinguish induction and competence for decarboxylation in combination with the analogue chemicals. The structural requirements for the substrates of the Pad-decarboxylation system were probed using a variety of sorbic and cinnamic acid analogues. High decarboxylation activity, ~100% conversion of 1mM substrates, required a mono-carboxylic acid with an alkenyl double bond in the trans (E)-configuration at position C2, further unsaturation at C4, and an overall molecular length between 6.5Å and 9Å. Polar groups on the phenyl ring of cinnamic acid abolished activity (no conversion). Furthermore, several compounds were shown to block Pad-decarboxylation. These compounds, primarily aldehyde analogues of active substrates, may serve to reduce food spoilage by moulds such as A. niger. The possible ecological role of Pad-decarboxylation of spore self-inhibitors is unlikely and the most probable role for Pad-decarboxylation is to remove cinnamic acid-type inhibitors from plant material and allow uninhibited germination and growth of mould spores.

AIMS:To assess whether assimilation tests in isolation remain a valid method of identification of yeasts, when applied to a wide range of environmental and spoilage isolates. METHODS AND RESULTS:Seventy-one yeast strains were isolated from a soft drinks factory. These were identified using assimilation tests and by D1/D2 rDNA sequencing. When compared to sequencing, assimilation test identifications (MicroLog™) were 18·3% correct, a further 14·1% correct within the genus and 67·6% were incorrectly identified. The majority of the latter could be attributed to the rise in newly reported yeast species. CONCLUSIONS:Assimilation tests alone are unreliable as a universal means of yeast identification, because of numerous new species, variability of strains and increasing coincidence of assimilation profiles. Assimilation tests still have a useful role in the identification of common species, such as the majority of clinical isolates. SIGNIFICANCE AND IMPACT OF THE STUDY:It is probable, based on these results, that many yeast identifications reported in older literature are incorrect. This emphasizes the crucial need for accurate identification in present and future publications.